Project Summary (Abstract) Cytochrome P450 (P450) enzymes are of considerable interest in the metabolism of chemical carcinogens, drugs, steroids, and fat-soluble vitamins. Research in these fields has had considerable impact in the field of drug development and also in medical practice, particularly endocrinology. Although P450 can now be considered a mature field, the current knowledge base and the availability of new technical approaches provide great opportunities to address several questions, which have great relevance in several areas of health. (A) Major effort continues in this laboratory to characterize reactions catalyzed by the ?orphan? P450s, i.e. those P450s that have only recently been described and have only limited information regarding the reactions that they catalyze. We propose to continue to use metabolomic/mass spectrometry methods we have initiated, focusing on human P450s 2S1, 4X1, 4Z1, and 20A1 in the next grant period, as well as any others we have resources to use or opportunities to pursue. (B) We continue to address questions of how P450s catalyze reactions. Two major questions will be addressed here: (i) Which form of oxygenated P450, FeO3+ or Fe(II)O2, is involved in C-C bond scission and C-C bond coupling, and (ii) how does desaturation occur? In the first question, we will study human P450s 17A1, 11A1, and 51A1, using 18O labeling methods we successfully employed with P450 19A1. Regarding C-C bond formation, we will use a bacterial P450 (158A2) and authentic FeO3+ (Compound I) in a collaborative study. Recently a ?pure? desaturase has been identified, P450 27C1, and we propose to study the retinol desaturation using kinetic isotope effects and other methods. (C) Sequential reactions are very common among P450s and can be either ?processive? or ?distributive,? depending on the extent to which the intermediate product dissociates and re-binds prior to the next catalytic step. Our research has shown variability in processivity among (human) P450s, still without a straightforward explanation in many cases. We propose to characterize the processivity with several human P450s, particularly 2E1, 2A6, 11A1, 11B2, and 51A1. Processivity is important for at least two reasons, one being that the release of potentially reactive products can be involved, and the other being that (in the case of drug targets) drugs can only be used to successfully inhibit one of the steps in a distributive reaction. Collectively, research in these areas is important in continuing to understand P450 reactions and to apply this to problems in health, particularly in the areas of drug development, chemical carcinogenesis, endocrinology, and nutrition.